Automated Phosphopeptide Enrichment for Gram-Positive Bacteria

Abstract

Protein phosphorylation in prokaryotes has gained more attention in recent years as several studies linked it to regulatory and signaling functions, indicating importance similar to protein phosphorylation in eukaryotes. Studies on bacterial phosphorylation have so far been conducted using manual or HPLC-supported phosphopeptide enrichment, whereas automation of phosphopeptide enrichment has been established in eukaryotes, allowing for high-throughput sampling. To facilitate the prospect of studying bacterial phosphorylation on a systems level, we here established an automated Ser/Thr/Tyr phosphopeptide enrichment workflow on the Agilent AssayMap platform. We present optimized buffer conditions for TiO 2 and Fe(III)-NTA-IMAC cartridge-based enrichment and the most advantageous, species-specific loading amounts for Streptococcus pyogenes , Listeria monocytogenes , and Bacillus subtilis . For higher sample amounts (≥250 μg), we observed superior performance of the Fe(III)-NTA cartridges, whereas for lower sample amounts (≤100 μg), TiO 2 -based enrichment is equally efficient. Both cartridges largely enriched the same set of phosphopeptides, suggesting no improvement of peptide yield by the complementary use of the two cartridges. Our data represent, to the best of our knowledge, the largest phosphoproteome identified in a single study for each of these bacteria.

In-Depth Characterization of the Clostridioides difficile Phosphoproteome to Identify Ser/Thr Kinase Substrates

“…The average abundances of pS and pT is 77% and 18%, respectively, and as expected, pY are less abundant (5%) ( Fig. 1 A and supplemental Table S3 ) in agreement with the distribution observed in C. difficile at stationary phase ( 22 ) and similar to the distributions found in B. subtilis and E. coli ( 14 , 20 , 50 ) ( Fig. 1 A ).…”

“…Known kinase substrates were also identified: (i) the anti–anti sigma factor RsbV on S57 as previously observed ( 22 , 51 ), likely by the anti–sigma and kinase factor, RsbW, which was also phosphorylated on S89; (ii) the HPr protein of the PTS on S45 in agreement with data obtained in vivo ( 22 ), and in vitro using purified HPr and HPr-kinase proteins ( 52 ) and on S11 as found in B. subtilis ( 22 , 53 , 54 ). As previously observed in other firmicutes ( 20 ), we found that the phosphoglutamine mutase, GlmM, which is involved in PG biosynthesis, was phosphorylated on S100 located at its active site and that the transition phase regulator, CodY, was phosphorylated on several residues including S216 shown as phosphorylated in Bacilli ( 55 ). Interestingly, toxin A, TcdA, is phosphorylated on multiple S residues ( supplemental Table S3 ).…”

“…1 Overview of the Clostridioides difficile 630Δ erm phosphoproteome. A , distribution of phosphorylated residues across all phosphoproteins found in C. difficile compared with other Gram-positives ( Bacillus subtilis , Streptococcus pneumoniae , Streptococuus pyogenes , and Listeria monocytogenes ) and Gram-negative ( Escherichia coli ) bacteria ( 14 , 20 , 50 , 77 ). pS means phosphoserine, pT phosphothreonine, and pY phosphotyrosine.…”

“…Several large-scale phosphoproteome analyses using large amounts of proteins have been reported in bacteria, including Escherichia coli ( 11 ), Bacillus subtilis ( 12 , 13 ), Streptococcus pneumoniae ( 14 ), Acinetobacter baumanii ( 15 , 16 ), and Streptococcus thermophilus ( 17 ). Recent improvements in phosphopeptide enrichment, liquid chromatography, and MS have increased the sensitivity of detection of phosphosites in bacteria ( 18 , 19 , 20 ). The phosphorylated proteins identified by phosphoproteomic studies are rather diverse among the tested firmicutes and, in general, a low overlap for STK targets is reported ( 18 , 19 ).…”

Automated liquid-handling operations for robust, resilient, and efficient bio-based laboratory practices

“…Miniaturized columns have not only allowed to accelerate the discovery of adequate purification conditions for pharmaceutical products [21,33], but also to identify key bottlenecks and obstacles before performing large-scale experimentation [34]. Cartridge-based purification has also been applied for solid-phase extraction [33], enrichment of phosphorylated proteins [35] and quantification of metabolites in clinical samples [36].…”